摘要
[目的]研究Cdh1和Num1之间是否存在相互作用,构建用于Cdh1和Num1免疫共沉淀试验的过表达菌株。[方法]首先在酵母菌株YWL630中用GAL1和3HA对CDH1进行标记,构建GAL1-3HA-CDH1NUM1-GFP菌株,再利用酵母四分体解离的方法使菌株YWL63和YWL490分别与该菌株杂交并进行四分体解离,从而得到所需菌株。[结果]成功构建不同基因型和配型的重组酵母菌株,其中YT52成功过表达了约340kD的Num1,YT53成功过表达了340kD的Num1和75kD的Cdh1,YT57成功过表达了75kD的Cdh1。[结论]经过同源重组的方法成功标记CDH1并诱导蛋白过表达,通过酵母四分体解离的方法构建用于Cdh1和Num1互作试验的过表达菌株,为揭示APC/C是否介导Num1的降解及机制奠定基础。
[Objective] To study whether there is an interaction between Cdh1 and Num1, and construct an over-expression strain for Cdh1 and Num1 co-immunoprecipitation experiments.[Method] Firstly, CDH1 was labeled with GAL1 and 3HA in yeast strain YWL630 to construct GAL1-3HA-CDH1 NUM1-GFP strain, and then yeast YWL63 and YWL490 were hybridized with the strain by yeast tetrad dissection method. The tetrad dissection was carried out to obtain the desired strain.[Result] The recombinant yeast strains with different genotypes and mating types were successfully constructed. YT52 successfully over-expressed Num1 with a size of about 340 kD. YT53 successfully over-expressed 340 kD Num1 and 75 kD Cdh1, and YT57 successfully over-expressed 75 kD of Cdh1.[Conclusion]The method of homologous recombination successfully labeled CDH1 and induced protein over-expression, and constructed an over-expression strain for Cdh1 and Num1 interaction experiments by yeast tetrad dissection thereby laying the foundation for revealing whether APC/C mediates the degradation of Num1 and its mechanism.
作者
李巧巧
庞文颖
海力
唐仙英
LI Qiao-qiao;PANG Wen-ying;HAI Li(Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China,Key Lab for Biotechnology of State Ethnic Affairs Commission,College of Life Science,South-Central University for Nationalities,Wuhan,Hubei 430074)
出处
《安徽农业科学》
CAS
2019年第10期1-4,9,共5页
Journal of Anhui Agricultural Sciences
基金
国家自然科学基金项目(31371366,31401155)
中南民族大学基本科研业务费专项资金项目(3212018CZY18021)
